TLR4 signaling must be tightly regulated to provide both effective immune protection and avoid inflammation-induced pathology. Thus, the mechanisms that negatively regulate the TLR4-triggered inflammatory response are of particular importance. Glia maturation factor-&#947; (GMFG), a novel actin depolymerization factor/cofilin superfamily protein that is expressed in inflammatory cells, has been implicated in mediating neutrophil and T cell migration, but its function in macrophage immune response remains unclear. In the current study, the role of GMFG in the LPS-induced TLR4-signaling pathway was investigated in THP-1 macrophages and human primary macrophages. LPS stimulation of macrophages decreased GMFG mRNA and protein expression. We show that GMFG negatively regulates LPS-induced activation of NF-&#954;B-, MAPK-, and IRF3-signaling pathways and subsequent production of proinflammatory cytokines and type I IFN in human macrophages. We found that endogenous GMFG localized within early and late endosomes. GMFG knockdown delayed LPS-induced TLR4 internalization and caused prolonged TLR4 retention at the early endosome, suggesting that TLR4 transport from early to late endosomes is interrupted, which may contribute to enhanced LPS-induced TLR4 signaling. Taken together, our findings suggest that GMFG functions as a negative regulator of TLR4 signaling by facilitating TLR4 endocytic trafficking in macrophages.